Screening of marine cyanobacteria for high palmitoleic acid production

FEMS Microbiology

Letters I33 t 1995) 137- 141

Screening of marine cyanobacteria for high palmitoleic acid production Tadashi Matsunaga a,*, Haruko Takeyama a, Yuki Miura a, Takeshi Yamazaki a, Hiroyuki Furuya b, Koji Sode a a Department of Biotechnology. Tokyo Unirencsityof Agriculture und Technology, Koganei, Tokyo 184. Japan h Yumamoto MSG. Co. Ltd. R&D Technical Lab., 118 Kamiymaguchi, Received 28 May 1995; revised 3 August

1995: accepted

Tokorozawa, Saitama 359, Japan IO September

1995

Abstract Screening of fatty acid composition in 150 strains of marine microalgae, cyanobacteria and green algae was carried out, and 20 strains showed relatively high contents of palmitoleic acid. Among them, two cyanobactetia, Phormidium sp. NKBG 041105 and Oscillatoriu sp. NKBG 091600, showed an unusually high cis-palmitoleic acid content (54.5% and 54.4% of total fatty acid, respectively). Phormidium sp. NKBG 041105 had the highest cis-palmitoleic acid content per biomass (46.3 mg (g dry cell weight)- ‘1, and cis-palmitoleic acid composition was found to be constant with varying temperature. These results indicate that this cyanobacterium could be considered as a new source for palmitoleic acid. Keywrds:

Marine cyanobacteria;

Phormidium sp.: Fatty acid; Palmitoleic

1. Introduction Novel bioprocesses utilizing marine microorganisms are expected to be key technologies for future bioindustry. Marine microalgae require carbon dioxide, sunlight and essential minerals in seawater for their cultivation. Screening these microalgae on their potential to produce useful materials enables the establishment of clean and economic bioprocesses [l-4]. We have previously reported the production of y-linolenic acid by the marine green algae, Chlorella sp. ISI, and the production of glutamate [61, cytokinin-like compounds [7,8] and a UV-A absorbing substance [9] by marine cyanobacteria.

= Corresponding author. [email protected]

Fax:

+8l

Federation of European Microbiological SSDI 037%1097(95)00350-9

(423) 85 77 13; E-mail:

Societies

acid

Recent pharmaceutical interest and current availability of polyunsaturated fatty acids have triggered the search for sources of their valuable compounds. Since monounsaturated fatty acids such as palmitoleic acid (Cl 6: l), undecylenic acid (Cl 1: I) and tridecenic acid ((213: I> have the potential for preventing brain disease, cerebra- and cardiovascular disease, and for enhancing the function of vascular smooth muscle cells [IO], they are expected to find novel and valuable uses in human nutrition and medicine. However, the availability of these monounsaturated fatty acids is limited. Although palmitoleic acid is present in some seed oils, its supply is insufficient with respect to the expected future demand, and in addition it is difficult to isolate palmitoleic acid from seed oils. Therefore, novel sources of palmitoleic acid have to be found.

138

T. Matsunaga et al./ FEMS Microbiology Letters 133 (19951 137-141

We have carried out primary screening of fatty acid composition in 150 strains of marine microalgae. In this study we report the screening of microalgae, especially cyanobacteria, which produce large amounts of palmitoleic acid.

The stereochemical conformation of palmitoleic acid was determined using a capillary column (ULBON HR-SS-10, 25 m X 0.25 mm i.d.; Shinwa Chemical Industries, Kyoto, Japan), using helium as the carrier gas and a temperature increasing from 180°C to 220°C at a rate of 2°C mini ‘.

2. Materials and methods

2.3. Lipid extraction and analysis

2.1. Organisms and culture conditions

The total lipid content was extracted with chloroform: methanol (2: 1, v/v) and purified according to Folch-Pibn et al. [ 111. The lipid extracts were analysed by TLC on silica gel plates. Total lipid classes were separated using acetone:benzene:water (9 1:30:8, v/v/v.). The lipid classes were detected with iodine vapor. Individual lipid classes were identified by their Rf values using known standards and specific spray reagents. Anthron regent was used for detection of glycolipids and Dittmer’s regent was used for phospholipids [ 121. Fatty acids of individual lipid classes separated by TLC were subjected to methanolysis and analysed by gas-chromatography as described above.

All marine microalgae were collected from the coastal areas of Japan and isolated in our laboratory. Algal cultures were axenic. The microalgal strains (Table 1) are being maintained in our laboratory and most are available from the authors upon request. Cultures (500 ml) were grown on BG-11 (marine) medium (ATCC catalogue, medium no. 617, containing 0.5 M NaCl), in flat-bottomed 1 liter flasks at a light intensity of 150 mmol quanta m-’ s-’ at 25°C with shaking. Algal cells grown exponentially for at least six days were harvested by centrifugation (8,000 X g) and used in fatty acid analysis. 2.2. Fatg acid analysis

3. Results and discussion Freeze-dried samples of microalgae (ca. 50 mg) were treated with 2 ml of methanol-HCl (19: 1) and C15:O (absence of C15:O in the sample was previously checked) as internal standard, and the mixtures were sealed in vials and heated to 100°C for 1 h. After the vials had cooled down, the samples were diluted with 1 ml H,O and extracted with 1 ml of n-hexane. The hexane layer was separated, dried and the residue was redissolved in hexane. Resulting methyl esters of fatty acids were recovered with n-hexane and analysed by gas chromatography using a stainless steel column (2 m X 3 mm i.d.1 packed with 15% (w/w) DEGS on Uniport B and operated isothermally at 180°C with nitrogen as the carrier gas. Sample analysis was performed in triplicate. Individual fatty acids were identified by comparing their retention times with those of standard samples. The quantities of fatty acids were estimated from the peak area on the chromatogram using the internal standard. Reproductivity was good (standard deviation 15%).

3.1. Screening microalgae

of palmitoleic acid producing marine

The fatty acid compositions of 150 microalgal strains (60 strains of cyanobacteria; Synechococcus, Synechocystis, Gloeothece, Chamaesiphon, Dermocarpa, Xenococcus, Anabaena, Pseudanabaena, Oscillatoria, Phormidium, LPP group and unidentified strains, and 90 strains of Chlorophyceae Chlorella and unidentified strains) were analysed. Twenty representative strains which showed relatively high contents of palmitoleic acid are presented in Table I. The predominant fatty acids in these algae were palmitic and oleic acid. The major fatty acids in the green algae examined were 16:0, 18: 1 and 18:2. These green algae contained 2 to 5% palmitoleic acid. Similar distributions have been reported in other Chlorophyceae [ 13,141. In contrast, larger contents of palmitoleic acid were observed in many cyanobacterial strains.

23

47

0 12.7 0 1.91

0 41.4 6.0 1.1 0.7 14.8 12.7

I .o

30

54

0 0 0 9.2

0 0 0 4.6

0 0 0 4.3

0 0 0 6.7

38

Tr Tr 0 26.2 54.4 0 0.4 6.1 0

0.8 2.3 2.0 38.7 4.4 5.8 2.8 22.8 4.2

0.9 I .4 2.0 46.4 8.6 3.2 Tr 23.7 8.9

1.3 I .9 1.7 29.9 13.5 4.8 1.3 30.9 7.8

0.6

NKBG 091600

NKBG 031401

NKBG 031500

NKBG 031504

NKBG 0203

Osc h

a

LPP

Cyanobacterium

of marine microalgae

85

0 0 0 9.7

1.4 0.9 Tr 20.3 54.5 0.3 8.0 Tr 0.3

NKBG 041105

Pho h

0 0 15.6 0.6 83

0 18.0 0 0.9 32

23.5 4.3 8.3 8.7 6.5 30.9

0.9 0. I

0.3

NKBG 040101

Xen b

1.5 Tr 45.6 8.0 0 2.3 16.9 6.6

Tr

NKBG 040601a

Cham

57

73

0.7 Tr 18.0 4.3 10.8 12.4 2.4 22.6 0 26.7 0 1.3

0.7

NKBG 0102b

43

17.9 Tr 32.6 29.2 1.7 2.5 5.9 0.6 0.1 0 0 4.0

0.7

NKBG 031301

Syn h

55

Tr 17.4 Tr 41.0 22.9 0 2.3 0. I 0 0 0 0 16.3 31

0 1.1 Tr 45.9 10.0 1.8 4.8 19.6 12.0 0 1.7 0 2.9

NKBG 040607

40

0.6 Tr Tr 42.5 8.9 0.9 22.8 Il.4 0 0 8.0 0 4.9

NKBG 041302

sp.; Xen, Xmocucc~s

NKBG 042704

sp.: Cham, Charnaesiphon

0.6 0.1 25.8 4.8 14.6 3.9 9.5 32.7 0 7.2 0 0. I

0.6

NKBG 032801

Der

Lpp, Lpp group; Osc, Ckillaroria sp.; Pho, Phomidiurn sp.; Glee, Gloeothecr sp.; Chl, Chlorelln sp, 18:3(y); 18:3 (w6), 18:3(cu); 18:3(w3), TFA; Total fatty acid. ’ Values are weight LTC of total fatty acid. h The strains are available from the authors upon request. ’ Values are mg (g dry cell weight)-’

TFA’

18:3(y) 18:3((Y) 20:o Others

12:o 14:o l4:l 16:O 16:l l6:2 18:O l8:l 18:2

Fatty acids

Table 1 Fatty acid composition

25

47

I.1 27.7 5.21 I .o I.0 0 1.6

I .o

0.1 0.7 Tr 31.6 10.9

NKBG 041001

45

Tr 0.2 0.4 41.8 4.6 5.31 I.3 25.8 8.72 1.5 0 0 0

NKG 041605

Chl b

128

0.6 I .4 Tr 16.1 4.5 0.4 I 3.7 29.6 4.92 0 8.2 0 0. I

NKG 040401

Green alga

73

0.I

0.8 0.9 Tr 25.0 4.2 1.91 7.7 6.7 9.3 0 0 13.1

NKG 040202

74

1.5

0 1.1 22.1 4.0 2.2 9.2 5.9 24.7 0 18.0 0

I .o

NKG 0102a

sp.; Der, Drmwcc~rpc~ sp.; Syn. Swrchococcus

0.3 2.5 Tr 55.6 5.4 0.8 3.2 16.2 4.21 0 8.6 0 2.7

NKBG 041905

Glee

140

T. Matsunaga

Table 2 Fatty acid compositions Rf value

of lipid classes from Phormidium

Lipid

0.93 0.89 0.77 0.74 0.6 I 0.44 0.18

et al. / FEMS Microbiology

Ratio(‘%/TL

Pigments TG GL CL CL DGDG PC

36.4

=)

7.8 9.5 19.7 I .2 1.7 23.4

l6:O

16:l

16:2

18:O

l8:l

I8:2

l8:3

75.0 23.8 0.9 6.8 8. I 5.9 0.4

IO.1 32.3 38.1 18.7 46.8 21.0 94.7

8.0 28.5 s3. I 71.3 45.2 65.7 2.5

0.2 Tr Tr Tr Tr 3.0 Tr

2.7 0 0 0 0 0 0

2.8 13.7 0.3 0.3 0 I.5 0.7

0.6 1.3 7.6 2.5 0 2.9 1.7

0.7 0.5 0 0.4 0 0 0

Among the cyanobacteria, two strains, Phormidium sp. NKBG 041105 and Oscillatoria sp. NKBG 091600, showed an unusually high palmitoleic acid content (54.5% and 54.4% of the total fatty acid respectively). The palmitoleic acid produced by these strains was confirmed as cis- 16: 1, the type of palmitoleic acid known to be produced by other cyanobacteria [ 151. These two virtually lacked 16:2 and 18:2, which are relatively abundant in other cyanobacteria. Other filamentous cyanobacteria showed palmitoleic acid ratios of 5-lo%, similar to that of unicellular cyanobacteria except NKBG 031301 and NKBG 042704. The highest cis-palmitoleic acid content per biomass (46.3 mg (g dry cell weight)- ‘) was found in Phormidium sp. NKBG 041 105, followed by Oscillatoria sp. NKBG 091600 (29.4 mg (g dry cell weight)- ’ ). Two Synechococcus strains (NKBG 042704 and NKBG 031301) contained much less palmitoleic acid (12.6 mg (g dry cell weight)-’ for both strains). Thus, Phormidium sp. NKBG 041105

Phormidium

sp.

NKBG 041105 Oscillatoria

sp. NKBG 09 1600

* Including

on fatty acid composition Growth temperature

Strains

(%/TFA)

< l4:O

TL. total lipid; TFA, total fatty acid; TG, triacylglycerol; CL, glycolipid: I Percentage levels estimated by weight of each lipid fraction,

Table 3 Effect of growth temperature

133 (1495) 137-141

sp. NKBG 041105

Fatty acid composition

Class

Letters

PC)

20 25 30 20 25 30

14:0, 14:l. 18:2, l8:3 and some unidentified

DGDG, digalactosyl

diacylglycerol:

may be the most suitable production.

PC, phosphatidyl

choline.

strain for palmitoleic

acid

3.2. Analysis of lipid classes The lipid composition of Phormidium sp. NKBG 041 105 was studied in further detail. The total lipid extracts were separated into 7 classes by their Rf value: a pigment, a neutral lipid, 4 glycolipids and a phospholipid. The relative amounts and fatty acid compositions of these lipid classes are presented in Table 2. The pigment appeared to be the major lipid (36.4%). One class of glycolipid (Rf: 0.74) was particularly rich in cis-palmitoleic acid (up to 71%). 3.3. EfSect of growth temperature acid composition

on cis-pulmitoleic

Since it has been reported that the proportion of polyunsaturated fatty acids decreases with increasing growth temperature [ 161, we investigated the effect

of Phormidium

sp. NKBG 041105 and Osci~~latoria sp. NKBG 091600

Main fatty acid composition

(‘%/TFA)

I6:O

16:l

18:O

Others *

20 21 21 23 23 30

55 54 60 57 43 49

8 8 2 3 I 3

I7 I7 I7 I7 33 18

fatty acids.

of growth temperature on the cis-palmitoleic acid composition of two strains. Phormidium NKBG 041 105 and Oscillutoria sp. NKBG 091600, cultivated at 20, 25 and 30°C (Table 3). The relative amount of cis-palmitoleic acid was not affected by change in temperature. The cellular contents of total fatty acid in each strain were similar at all growth temperatures tested. Insensitivity of fatty acid compositions to temperature is very important considering a simple system for commercial production of fatty acids. Therefore. these algae are likely to be suitable for fatty acid production by outdoor mass culture. where growth temperature varies according to the weather. In conclusion, we have succeeded in identifying two marine cyanobacteria, Phormidium NKBG 041 105 and Oscillatoria sp. NKBG 09 1600, with a high cis-palmitoleic acid content. independent of temperature in the range of 20-30°C. These cyanobacteria could be a new source for the production of ci.s-palmitoleic acid.

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